Woody biomass can be employed as a sustainable source of energy and is a valuable alternative to fossil fuels. More specifically, the biorefining of lignocellulosic material into fuel ethanol and lignin materials has the potential to displace a portion of petrol and oil based materials. It is likely that, with the depletion of global oil reserves and increasing awareness of the environmental and national security issues associated with dependence on fossil fuel, biomass will become a key resource for the production of transport fuel in much of the world.
The conversion of lignocellulosic biomass into fuel ethanol may offer the ideal solution given the rapid growth of short rotation crops such as shrub willow (Salix spp.).
Two of the main components of wood, cellulose and hemicellulose, are polymers of simple sugars that can be converted into ethanol and/or other chemicals by fermentation. This ethanol can be used as a transportation fuel either on its own or as an ethanol-gasoline blend. Ethanol-gasoline blends of up to 10% ethanol can be used without any engine modification or loss in engine performance (Hunt, V. D. (1981) The Gasohol Handbook, New York, Industrial Press). Lignin, the third main component of wood, is a potential raw material for the production of plastics, adhesives and resins (Lora and Glasser (2002) J. Polymers Environ. 10:39-47). The use of lignin in high value products, rather than as boiler fuel, will off-set the high costs traditionally associated with the processing of wood and production of ethanol.
Willow biomass plantations can be easily and efficiently established from dormant stem cuttings using mechanical systems. Shrub willows respond to coppicing after the first growing season by prolific production of new stem growth in the second growing season. Above ground woody biomass is harvested during the dormant season. During the spring following each harvest, the remaining portion of the willow plant, known as the stool, responds by producing numerous new stems, initiating a new cycle of growth that can be harvested in another two to four years. This cycle can be repeated for six to eight harvests before the stools need to be replaced.
Lignocellulose is a complex substrate composed of a mixture of carbohydrate polymers (namely cellulose and hemicellulose) and lignin. The conversion of lignocellulosic biomass into ethanol relies mainly on the efficient separation of these cell wall components to allow the hydrolysis of the carbohydrates polymer into fermentable sugars. Most of the processes using high temperature or pressure with acid, caustic or organic solvent are able to provide a cellulose substrate that can be chemically or enzymatically converted into fermentable glucose (Wyman et al. (2005) Bioresource Technology 96:2026-2032; Mosier et al. (2005)Bioresource Technology 96:673-86). In general, the yield and hydrolysis rate of cellulose increases when biomass is fractionated under conditions of high temperature and extremes of pH. Under these severe conditions, however, the overall carbohydrate recovery is often compromised due to extensive degradation of the hemicellulose sugars (mainly xylose in hardwood), which comprise a significant fraction of the lignocellulosic feedstock (hardwood: Rughani and McGinnis (1989) Biotechnol. Bioeng. 33:681-686; Bakker et al. “Biofuel production from acid-impregnated willow and switchgrass”; 2nd World Conference on Biomass for Energy, Industry and Climate Protection, 10-14 May 2004, Rome, Italy; Li et al. (2005) Appl. Biochem. Biotechnol. 125:175-88; Sassner et al. (2005) Appl. Biochem. Biotechnol. 121-124:1101-17; Pan et al. (2005) Biotechnol. Bioeng. 90:473-81; softwood: Boussaid et al. (1999) Biotechnol. Bioeng. 64:284-9; Yang and Wyman (2004) Bioresource Technol. 86:88-95; Knauf and Moniruzzaman (2004) Intl. Sugar J. 106:147-50; Mosier et al. (2005) Ibid). Also, the degradation products generated by extensive hydrolysis (phenol, furans and carboxylic acid) can potentially inhibit further fermentation steps (Palmquist et al. (1999) Biotechnol. Bioeng. 63(1):46-55; Klinke et al. (2004) Appl. Microbiol. Biotechnol. 66:10-26). Furthermore, severe pre-treatment conditions, including the use of acid catalysts, can chemically alter the nature of the recovered lignin. A consequence of this is a decrease in the suitability of the lignin for some high value applications (Lignin Institute Dialogue Newsletter (2001) 9(1); Lora and Glasser (2002) Ibid; Matsushita and Yasuda (2003) J. Wood Sci. 49:166-171).
When water is used as the sole fractionation agent, the majority of the hemicellulose sugars can be recovered through autohydrolysis (Garrote and Parajo (2002) Wood Science Technol. 36:111-123). However, due to inefficient delignification, this maximization of the hemicellulose sugar yield is usually done at the expense of the cellulose/glucose enzymatic conversion (Negro et al. (2003) Appl. Biochem. Biotechnol. 105:87-100; Chung et al. (2005) Appl. Biochem. Biotechnol. 121:947-961; Kim and Lee (2006) Bioresource Biotechnol. 97:224-232). Use of a second stage oxidative treatment was shown to improve the cellulose/sucrose conversion following the hot water treatment but not always as a result of efficient lignin removal (Brownell and Saddler (1987) Biotechnol. Bioeng. 29:228-35; Wyman et al. (2005) Bioresource Technol. 96:1959-1966; Kim and Holtzapple (2006) Bioresource Technol. 97:583-591).
The efficient removal of lignin under mild conditions can be achieved using the OrganoSolv™ process. This type of pre-treatment involves the use of an aqueous organic solvent, usually ethanol, to achieve the simultaneous removal of the hemicellulose sugar and lignin in separated streams. The cost associated with the use of an ethanol solvent is reduced by producing the ethanol on site and efficiently recycling it, as taught, for example, by U.S. Pat. No. 5,788,812. The conversion rate of the cellulose solid fraction provided by aqueous ethanol pre-treatment is mainly affected by the inefficient removal of the hemicellulose sugar when lower water/solvent ratios are used to maximize the lignin recovery (Holtzapple and Humphrey (1984) Biotechnol. Bioeng. 26:670-676; Chum et al., (1988) Biotechnol. Bioeng. 31:643-649). Increasing the water/ethanol ratio, or the addition of a chemical catalyst to the solvent, increases the hemicellulose sugar removal but is associated with a reduction of lignin removal and increased hemicellulose sugar degradation (Holtzapple and Humphrey (1984) Ibid; Rughani and McGinnis (1989) Ibid; Pan et al. (2005) Ibid).
Successful advancements in enzyme production technology have resulted in a lower cost of the hydrolytic enzyme required to obtain a high conversion rate of cellulose to glucose. However, because the enzymatic hydrolysis activity is strongly inhibited by the hydrolysis products (sucrose and short cellulose chains), simultaneous fermentation of the released sugar (SSF for simultaneous saccharification and fermentation) can greatly improve the overall cellulose/ethanol conversion using lower enzyme loading. Several technologies are now available that allow a broader use of the biomass at lower cost under a variety of less constraining conditions (reviewed in Lin and Tanaka (2006) Appl. Microbiol. Biotechnol. 69:627-642).
Whereas the fermentation of glucose can be carried out efficiently by a variety of organisms, the bioconversion of the pentose fraction (xylose and arabinose) presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize pentose and convert them to useful compounds, such as ethanol (reviewed in: Aristidou and Penttila (2000) Curr. Opin. Biotechnol. 11:187-198). Alternatively, the pentose fraction which is predominantly xylose in hardwood species such as Salix, can be recovered from the water stream and converted to xylitol for use as a valuable food product additive. By-product streams from this process (furfural, acetic acid, para-hydroxybenzoic acid and vanillin) may also be fractionated subject to market price. Furfural, the easiest by-product to market, can be obtained by distillation from the same fraction. The acetic acid may also be recovered to produce peroxyacetic acid for pulp.
Ethanol-soluble lignin is considered to be of higher value because of its ease of recovery and its suitability for a wide range of industrial applications compared with water-soluble lignin, such as that recovered from the Kraft process often employed by the pulp and paper industry. Extraction of Kraft lignin requires high volumes of solvent and has a narrower range of applications (Funaoka et al. (1995) Biotechnol. Bioeng. 46:545-552; Lora and Glasser (2002) J. Polymers Environ. 10:39-47; Kubo and Kadla (2004) Macromol. 37:6904-6911; Lawoko et al. (2005) Biomacromol. 6:3467-3473).
Lignin extracted using the OrganoSolv™ process differs significantly from that extracted via the Kraft process. OrganoSolv™ lignin has a molecular weight of 700 to 1550 g/mol, low polydispersity, a glass transition temperature of 70 to 170° C., a high relative amount of phenolic hydroxyl groups, and a low degree of chemical modification (Lora and Glasser (2002) Ibid; Kubo and Kadla (2004) Ibid; Lawoko et al. (2005) Ibid). This lignin can be used in the manufacture of molding compounds, urethane epoxy and formaldehyde resins, antioxidants and controlled-release agents. Ethanol-soluble lignin from hardwoods is recovered by diluting the aqueous ethanol pre-treatment effluent with water and acid to form a solution with a pH of 1.5 to 2.7 and an alcohol content of 30% (v/v) (or a ratio of aqueous-ethanol effluent to the acid water of 0.35 to 0.70). After drying, the precipitated lignin is obtained in the form of a powder (U.S. Pat. No. 5,788,812).
Acid catalyzed OrganoSolv™ pulping was originally developed by Theodor Kleinert as an environmentally preferred alternative to Kraft pulping (U.S. Pat. No. 3,585,104). It was later found that a variation of the operating conditions could very efficiently convert the lignocellulosic material to sugars and lignin. In the 1980s, a 16 liter continuous flow reactor pilot plant that processed bagasse to sugars was built (Dedini, Brazil). A concentrated solution of acetone with a small amount of acid was used to solubilize the lignocellulosic component of the bagasse (U.S. Pat. No. 4,409,032).
An OrganoSolv™ process using aqueous ethanol to produce a clean biofuel for turbine generators was developed by the University of Pennsylvania and the General Electric Company in the 1970s. Subsequent modification by the Canadian pulp and paper industry resulted in the Alcell™ pulping process (U.S. Pat. No. 4,100,016). The long-term economic viability of the Alcell™ process was significantly improved using technology for the recovery of lignin and furfural by-products from the organic pulping liquor (U.S. Pat. Nos. 4,764,596, 5,681,427 and 5,788,812). A commercial Alcell™ pulping plant processing metric tons of hardwood per day was established in 1989 in New Brunswick Canada. The plant was operated for several years but was eventually shut down due to external economic factors. More recently, a patent application was published relating to an integrated operation for processing sugarcane that combines the OrganoSolv™ Alcell™ process, pulping and fermentation to reduce the capital and operating cost by providing a high degree of internal process recycling (US Patent Publication No. US 2002/0069987).
There remains a need in the art for a process for producing ethanol from woody biomass which can be established at a relatively low cost and be profitable by maximizing the yield and recovery of valuable by products such as natural lignin and xylose.